Abstract Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome), and opthalmologic disorders (age related macular degeneration). Extensive studies have identified over 150 genes that regulate melanin production in human cells, but have not yet yielded a complete understanding of the pathogenesis of disorders of melanin production. Recently, we combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized small interfering RNAs to identify 94 novel regulators of melanogenesis in human cells. Secondary validation of gene targets identified using this functional genomics approach revealed that the approach had a low-false positive and off-target rate. Intriguingly, several components of the autophagy pathway were identified as novel regulators of melanin production in this analysis. Detailed validation of these putative autophagy regulators revealed that these genes impacted melanogenesis both in vitro and in vivo. In this proposal, we define the mechanism by which autophagy regulatory genes control pigment production in human skin. Initial studies will validate whether these putative autophagy regulators impact both melanin production and autophagy in MNT-1 cells and normal melanocytes. A defined approach will be utilized to determine the impact of depletion of autophagy regulators on the delivery of specific cargo to the melanosome. Biochemical approaches will be utilized to identify melanosome components that interact with novel autophagy regulators. Finally, a human skin equivalent model will be utilized to determine the contribution of individual autophagy regulators to melanin production in human skin. Through these studies, we hope to determine how the melanocyte utilizes the autophagy machinery to deliver specific protein cargo to the melanosome in vivo. PUBLIC HEALTH RELEVANCE: Project Narrative (relevance) Melanin protects the skin, eyes, and neurologic system from toxic insults and is aberrantly regulated in skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome) and opthalmologic disorders (age related macular degeneration). Genome-wide siRNAi-based functional genomics recently determined that several known and putative autophagy regulators impact melanogenesis in human cells. In this proposal, we determine how regulators of autophagy also impact melanin production. Through these studies we will gain insight into the regulation of vesicular trafficking in melanocytes and how these processes impact melanogenesis.